Tubercle and Lung Disease (1995) 76, 578-581

© 1995 Pearson Professional Ltd

Tubercleand
LungDisease

A simple method of DNA extraction from Mycobacterium tuberculosis

M. Jaber*, A. Rattan*, A. Verma*, J. TyagF, R. Kumar*

*Departments of Microbiology and ~Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New
Delhi, India

S U M M A R Y. The application of molecular biology methods has contributed significant insight into the epi-
demiology of infections caused by Mycobacterium tuberculosis. However, most of the techniques remain out
of reach of laboratories in the tropics. We describe here a simple method for extraction of DNA from the
pathogen, a first step in the application of molecular biology tools.

R/~ S U M/~. La mise en pratique des m6thodes de biologic mol6culaire a apport6 des apergus significatifs
quant h l'6pid6miologie des infections caus6es par Mycobacterium tuberculosis. Pourtant, la plupart des ces
techniques restent hors de port6e des laboratoires en r6gion tropicale. Est d6crite ici une m6thode simple
d'extraction de I'ADN du pathog~ne, premier pas dans l'application des outils apport6s par la biologic
mol6culaire.

R E S U M E N . La aplicaci6n de los m6todos de biologia molecular ha contribuido significativamente a

profundizar el conocimiento de la epidemiologia de las infecciones causadas por Mycobacterium tuberculosis.
Sin embargo, la mayoria de estas t6cnicas permanecen fuera del alcance de los laboratorios en las regiones
tropicales. En este articulo describimos un m6todo simple de extracci6n del ADN del pat6geno, primer paso en
la aplicaci6n de las herramientas de la biologia molecular.

INTRODUCTION tion of DNA from mycobacteria isolated on L6wenstein

With the resurgence of tuberculosis in developed
countries, there have been increased attempts to use
DNA technology for diagnosis and epidemiology of
the infection. An important first step in performing
these studies is to isolate the DNA from Mycobacterium
tuberculosis.
Various protocols have been used for the isolation
and purification of mycobacterial DNA. 1-9 The yield is
satisfactory and purity of the isolated DNA has been
reported to be of good quality and quantity, and suitable
for various DNA related techniques. However these pro-
tocols require culturing of mycobacteria in liquid media,
physical disruption such as lysis by French pressure
cell pretreatment of the bacilli with chemicals such
as cycloserine, followed by the addition of enzymes
(lysozyme and proteinase K) and use of a detergent, so-
dium dodecyl sulfate (SDS), to ensure the complete lysis
of the cell and the release of its DNA.
We report a simple and rapid method for the extrac-
Jensen (LJ) slant which can be easily applied in the
routine laboratory, thus avoiding the use of biochemicals
which are expensive and require storage at low tempera-
ture and physical equipment that is difficult to handle,
costly and mostly unavailable in mycobacteriology labo-
ratories in developing countries.
MATERIALS AND M E T H O D S
Bacterial strains
Standard strains
M. tuberculosis strains H37Rv and H37Ra were obtained
from our collection.
Clinical isolates
Seventy clinical isolates from patients with pulmonary
tuberculosis identified as M. tuberculosis on the basis of
typical characteristics were studied? °

Protocol of DNA extraction

Correspondenceto: Dr AshokRattan, Assoc.Prof.,Departmentof
Microbiology,AIIMS,AnsariNagar, New Delhi 110 029, India. 1. Transfer 1-2 loopfuls (60-120 mg) of mycobacte-

578
 DNA extractionfrom Mycobacterium tuberculosis 579

rial culture from an LJ slant into a screw-capped

glass tube containing 1 ml of lysis buffer (see below)
and three glass beads, vortex to suspend the bacte-
rial cells and transfer to an Eppendorf tube.
2. Incubate the tube at 4°C for 30 min.
3. Transfer immediately to a water bath set at 75°C for
30 min.
4. Centrifuge at 10 000 g at 4°C for 10 rain and trans-
fer the supernatant into another Eppendorf tube.
5. Precipitate the DNA in the supernatant with 0.5
volume of ice cold ethanol, keep at -20°C for
20 r a i n - 1 h.
6. Centrifuge at 12 000 g for 15 min and redissolve the
DNA pellet in 0.8 ml lysis buffer without Tween
80.
I[--
7. Extract the DNA first with phenol-chloroform (1:1,
v/v) and then with chloroform:isoamyl alcohol
(24:1, v/v).
8. Add 5 M NaC1 to a final concentration of 0.3 M.
9. Precipitate the DNA with 2 volumes ethanol,
keep for 20 rain or overnight at -20 °C, centrifuge at
12 000 g for 15 min.
10. Wash the DNA with 1 ml of 70% ice cold ethanol,
dry DNA pellet and dissolve in TE buffer pH 8.0
(60-120 gl).

Lysis buffer
6 M Guanidinium hydrochloride (sigma); 50 mM EDTA;
1 mM 2-mercaptoethanol; 0.05% Tween 80.
Fig. 1--Effect of ethanol precipitation on RNA after cell lysis. Lane
1: without ethanol precipitation;Lane 2: ethanol precipitation for 1 h;
Lane 3: ethanol precipitation overnight. Note that the amount of RNA
(-->) decreases with the increase in precipitation time at -20 °C.

Important notes
1. If no DNA precipitate is visible in step 5, skip step 6
and proceed to step 7.
2. Ethanol precipitation in step 5 is crucial to the isola-
tion of DNA which is contaminated with a minimal
amount of RNA. The amount of RNA decreases
significantly with the increase in precipitation time at
-20°C. Precipitation of the DNA for 20 rain to 1 h
usually eliminates most of the RNA. On the other
hand, omission of step 5 results in the recovery of a
large quantity of RNA. (Fig. 1).
3. The superuatant from step 7 can also be processed for
DNA extraction; at least 30% of the DNA remains in
the supernatant.
Southern blot hybridization
M. tuberculosis DNA was digested with PstI and probed
with 0.8 kb SmaI-Mlul fragment ofpAK52 derived from
M. tuberculosis DNA. ~
Comparison with other DNA extraction methods
We compared this method with the methods described
by Katoch and Cox, 2 Bose et al,8 and Rastogi et al9 with
five strains.
RESULTS AND DISCUSSION
In the present study we used the chaotropic agent
guanidinium hydrochloride. This agent inactivates
both RNase and DNase, dissociates nucleoprotein, and
disturbs cellular and subcellular structure, and its pH and
ionic strength favour the native form of the DNA? 2The
effect of this solvent and the thermal degradation of the
cellular organic material, combined with further protein
denaturation by phenol and chloroform, have resulted
in the isolation of biologically active and completely
intact DNA from 70 M. tuberculosis (clinical isolates)
and laboratory standard strains (H37Rv and H37Ra)
directly from LJ slant.
Spectrophotometric measurement for DNA was car-
ried out at OD 260 nm and OD 280 nm in a double beam
UV spectrophotometre (Shimadzu UV 160 A). The con-
centration of DNA was calculated from the absorbance
at OD 260 nm, and the level of purification was esti-
mated by the relative absorbance ratio. The yield of
DNA obtained from one gram of bacteria was 1.8 mg/G.
The yield of DNA extracted from 1 to 2 loopfuls of
bacteria (60 to 120 mg weight of bacteria) ranged from
84 to 278 [tg and the purity ranged from 1.84 to 2
(average 1.9) (Fig. 1),
580 Tubercle and Lung Disease

advantages of our method are that there is no need to

Fig. 2--Southern blot hybridization: Mycobacterium tuberculosis
DNA was digested with PstI and probed with 0.8 kb SmaI-MluI
fragment o f p A K 5 2 derived from M. tuberculosis DNA. 1° Lane 1:
H37Ra; Lane 2 and 9: H37Rv; Lane 3-8: clinical isolates of M.
tuberculosis.
subculture mycobacteria in liquid medium, it is rapid,
and it does not require any specialized equipment or
costly reagent.
Due to the small sample volume (1.5 ml), the whole
procedure was performed in an Eppendorf tube. Using
our protocol 12-14 samples can be processed at one
time in less than 4 h.
Guanidium thiocyanate together with silica particles
or diatoms have been used by Boom et al ~3 as a rapid
and simple method for purification of nucleic acid from
mammalian cells, viruses and gram negative bacteria.
However, although their protocol was suitable for purifi-
cation of nucleic acid from several pathogenic gram
negative bacteria, nucleic acid could not be purified
directly from gram positive bacteria.
Noeal et alia have used their protocol for the extrac-
tion of mycobacterial DNA for the PCR test. However,
simple extraction of mycobacterial DNA by boiling
in distilled water has been used successfully for the
PCR test. 15 But the quantity and the quality of the DNA
required for PCR is extremely small, when compared
to the requirement of DNA for RFLP and other finger-
printing techniques.
To conclude, the method described here is simple,
rapid, inexpensive and should enable laboratories in
developing countries with modest equipment to partici-
pate in multicentric studies to investigate the interna-
tional transmission of tuberculosis. 16

The quality of the DNA was assessed by Southern
blot hybridization of PstI restriction enzyme digests of
mycobacterial DNAs with a 0.8 kb fragment of M. tu-
berculosis. A hybridization signal of 2.8, 2.1, 1.35 and
0.75 kb was obtained, indicating that the DNA was of
satisfactory quality and integrity for use in hybridization
analysis (Fig. 2).
Different DNA extraction protocols for M. tuberculo-
sis rely on the use of either the physical or the enzymatic
method, which may or may not require pretreatment of
the bacilli with cell-wall modifying agents. Our protocol
was compared directly with a representative of each
type. 2,8,9 The Table lists the major features of the four
methods as well as the results obtained. The main
Acknowledgement
The authors with to acknowledge the support and advice provided
by Prof. P. N. Tandon, Prof. of Neurosurgery, AIIMS; Dr Rama
Mukheljee, Deputy Director, NII, Dr V. M. Katoch, Deputy Director,
JALMA and Dr Bimal K. Das, Dept of Microbiology, AIIMS during
the course of this work.
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Table. Direct comparison between four methods

Method Subculture Chemical Physical Time Cost (in Average yield Purity
into liquid pretreatment pretreatment in hours US $) of DNA in of DNA
medium of reagents* mg/G
Reported No No No 4 0.7 1.8 mg/G 1.9
protocol
Katoch Yes No Yes 48 3.0 1.6 mg/G 1.8
and Cox ~
Bose et al a Yes No No 6 7.0 1.5 mg/G 1.9
Rastogi Yes Yes No 75 16.0 1.0 mg/G 1.8
et al9
*The cost of reagents was calculated based on Sigma Catalogue 1994.

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